The genomic organization of the 4.6-Mbp human Major Histocompatibility Complex (MHC) will be best understood in a comparative evolutionary context. The order and gene content of the five subregions of the MHC (extended class II; classical class II; class III; classical class I; and extended class I) appear to be largely conserved between the MHCs of primates (human and rhesus macaque) and rodents (mouse and rat). The principal exception being the classical class I region of the MHC, where the genes encoding the antigen binding class I molecules reside in four distinct sites along this 1.8 Mbp region and display variation in size and gene number both within and between species. In order to determine the genomic organization and complete nucleotide sequence of the domestic cat (Felis catus) MHC, DNA probes for 61 markers (spanning ARE1 in the class II region to the olfactory receptor complex (OLFR) in the extended class I region) were designed from human MHC reference sequences and used to screen domestic cat genomic DNA large-insert libraries bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) libraries with an average insert size of 138 and 80 Kbp, respectively). State-of-the-art, high-throughput technologies were then applied to order these cloned genomic DNA fragments into a BAC/PAC contig map of the domestic cat MHC. Putative MHC positive BAC/PAC clones were arranged in a 96-well plate format and (a) rescreened by colony hybridization and (b) BAC/PAC DNAs were purified for fingerprint analysis and Southern hybridization. To generate contig maps of large insert clones, complete Hind III-digested BAC/PAC DNAs were fingerprinted by electrophoresis and the data analyzed using the Fingerprinting Contigs (FPC) software developed by C. Soderlund, which assembles a minimal tiling path of clones using an algorithm to cluster clones into contigs based on their probability of coincidence scores. Using these approaches, we constructed sequence-ready BAC/PAC contig maps of 2.85 Mbp containing class II-class III-class I (proximal region) and 0.50 Mbp of MHC class I/extended class I region of the domestic cat MHC containing in total 194 BAC and 18 PAC clones. Partial nucleotide sequencing confirmed that the two contigs did not overlap, suggesting that perhaps the genomic organization of the domestic cat MHC differs from the primate and rodent models. Therefore, eleven domestic cat BAC clones for four subregions of the mammalian MHC (classical class II, class III, class I, and extended-class I) were chosen for fluorescent in situ hybridization (FISH) to domestic cat chromosomes. All nine clones from the 2.85 Mbp contig hybridized to the pericentromeric region of cat chromosome B2 (FCAB2), whereas the two clones from the 0.50 Mbp contig hybridized to the telomeric region of the p-arm of B2. Previous studies had shown that cat chromosome B2 was homologous to human chromosome 6 (HSA6-note that the human MHC is located on the p-arm of HSA6) and comparative mapping using the domestic cat radiation hybrid panel had shown that FCAB2 had undergone two inversions relative to HSA6: one encompassing the distal q-arm of B2 and the other encompassing the entire p-arm of B2. Our results were consistent with the hypothesis that the breakpoint of the later inversion mapped to the MHC. To test this hypothesis, primer pairs for 13 loci from the 2.85 Mbp contig and 2 loci from the 0.50 Mbp contig were designed and screened against the 5000 Rad domestic cat radiation hybrid panel. The results not only confirmed that the p-arm of domestic cat B2 is inverted relative to human chromosome 6, but also demonstrated that one inversion breakpoint localized to the distal segment of the MHC class I between tripartite motif-containing 39 (TRIM39) and TRIM26 (this corresponds to the HLA-92 region of the human MHC). The inversion thus disjoined the 2.85 Mb of MHC containing class II-class III-class I (proximal region) from the 0.50 Mb of MHC class I/extended class I region, such that TRIM39 is adjacent to the chromosome B2 centromere and TRIM26 is adjacent to the B2 telomere in the domestic cat. In addition, no evidence for antigen binding class I genes in the HLA-A region or any other part of the 0.5.0 Mbp contig was obtained, but the region corresponding to the human HLA-B and HLA-C genes has undergone considerable expansion during evolutionary history and contains at least 12 class I genes/gene fragments similar to the mouse H2-Q region.